Function and Regulation of Cotransport in Man

O'Neill, W

Abstract

This award will provide the candidate, a clinical endocrinologist with considerable research experience, the opportunity to further his expertise in membrane biology and apply this expertise to the study of human disease. The long-term research plans of the candidate involve the development of a model system for studying membrane function in metabolic diseases. The research objective of this application is to determine the function and regulation of cotransport activity in man, and to identify the protein which mediates this transport process. These studies will be aided by our recent discovery of a patient with thyrotoxic periodic paralysis (TPP) in whom erythrocyte cotransport it absent. The kinetics of cotransport will be characterized in cultured human fibroblasts and the effect of T3, insulin, and epinephrine on this transport process will be determined. Fibroblasts from the TPP patient and normal fibroblasts treated with a cotransport-inhibitor will be compared to normal fibroblasts to determine the consequences of an absence of cotransport on cell volume, intracellular Na+ and K+ concentrations, membrane potential, and the response of the cells to changes in the cation concentration and osmolarity of the external environment. The effect of T3, insulin, and epinephrine on the above parameters in these cells will be compared to the effects in normal cells, in an attempt to understand the pathophysiology of TPP. Affinity chromatography with immobilized bumetanide, and immune precipitation with specific antiserum, of solubilized membranes from normal and TPP erythrocytes will be used to identify the cotransport protein and characterize the defect in the TPP erythrocytes. The candidate will be working within an established and productive membrane biology laboratory which has expertise in all facets of membrane biochemistry and biophysics, which will provide an excellent opportunity for the further development of the candidate in this area. The candidate will also hold an appointment in the Endocrine Division of the Department of Medicine, which will enable him to maintain clinical skills and allow for the opportunity to apply his research to clinical problems.